Chapter V

Black box growth

Growth without non-catabolic product

Single substrate limited growth

Hyperbolic kinetic equation for specific

substrate uptake rate (qS)
q max
S

-qS

0.5 qSmax

KS

( qS )

CS

qSm ax

CS
K S CS

KS: substrate affinity [kg substrate/m3]

qSmax: maximal uptake rate [kg S consumed/ kg X present-h]
3

Substrate uptake for maintenance

Some part of substrate is used for producing energy needed for

cells maintenance. This part is catabolized with a rate mS.
Hence, mS = kg substrate catabolized for maintenance / kg
biomass presenthour.

Herbert-Pirt relation for substrate

(qS )

m
)
S
max

YSX

It means the substrate taken into cells are used for growth and
maintenance only. Both mS and YSXmax are model parameters.

-qS

1
max
YSX

(-mS)

Herbert-Pirt linear plot for

substrate, non-catabolic
product is absent

Combination of the qS equation with HerbertPirt relation

- qSm ax CS

mS
K S CS

m ax
YSX

CS = 0 , = mSYSXmax = -kd
CS >>>, = [-qSmax-(mS)]YSXmax = max
= 0, (-qS) = (-mS), which occurs at CS = CSmin.
The specific growth rate equation based on single substrate is
then:
min

CS
K S CS

max CS

The plot of - CS
max

-kd

CSmin

m ax

For mS = 0,

CS
CS
K S CS

(Monod equation)
7

Using Herbert-Pirt relation to calculate other

rates
Example:

The case of aerobic growth on glucose, N-source is ammonia

Suppose qS = 0.3125 + 0.0015
Growth reaction:

-0.3125 C6H12O6 + aNH4+ + bO2 + cH+ + dH2O +

C1H1.8O0.5N0.2 + eCO2
Use the yield and elemental balance to obtain a, b, c, d, e
The catabolic reaction is: -C6H12O6 - 6O2 + 6CO2 + 6H2O
What are (-qNH4+), (-qO2), qCO2, qH+, qH2O?
8

How to measure the kinetic parameters?

Obtaining max and YSXmax.
Using batch fermentation at constant volume where CS >>
Hence = max (constant, during the exponential phase)
qS = qSmax (constant),
YSX = YSX max = (CX-CXo)/(CSo-CS)

Obtaining KS and mS.

Using chemostats, is the variable to get -qS. Apply HerbertPirt relation and plot qS vs. to get YSXmax and mS.
Plot qS vs. CS to obtain KS and qSmax.
9

Further reading
The Herbert-Pirt relation including non catabolic product
formation
Extended Herbert-Pirt relation
Different qP - function
Effect of temperature and pH.

10

Chapter VI
Growth and product formation in
bioreactors

11

Fermentation processes
substrate

fermenter

Downstream
processing

products

wastes
The upstream processing costs about 20 50 %, whereas the
downstream processing costs about 50-80 %

12

The type of reactors and processes

The reactors systems:
Aerated stirred tank reactor 0.1 1000 m3 with cooling
equipment
Bubble column 0.1 10,000 m3 with airlift and cooling
equipment
Bioreactors for immobilized cells/biocatalysts (packed bed,
fluidized bed, trickle bed)
The types of processes used are batch, continuous, and fed
batch systems. Mostly, batch and fed batch reactors are used in
industry whereas continuous reactors (chemostats) are used in
laboratories.
13

Fermenter utilities
Sterilization:
1. Autoclaving

2. Live steam batch

3. Membrane
4. Heat shock for continuous system (less odor, energy,
degradation, dilution, time)
Mixing by stirrer
mix 20 200 seconds, power 1-3 kW/m3.
Oxygen input
50 200 mol/m3-h.
14

Fermenter utilities (contd)

Air filtration, using membrane / bed filter with dry air about 60
m3 air/m3 broth-h.

Compressor, 2 bar, 150oC.

Heat removal

Cooling jacket

Coil

With cool water temperature at 10oC and capacity is about

50,000 kJ/m3 reactor-h.

15

Laboratory chemostat and industrial fermenter

Applikon laboratory chemostat at

Kluyverlab, Delft University of
Technology

Labatt Breweries - London, Ontario

New Vertical Fermenter
16

The world largest fermenter

There may be biological waste treatment in larger vessels, but the world's largest
fermenter is shown in these photos taken from Chemical and Engineering News. The
fermenter is 200' high and 25 ft diam. The first photo (Chem. Eng. News, 10-Apr-78)
shows the fermenter being transported on vehicles with tank treads.

17

Packed bed fermenter

18

What do we want?
High yield of biomass
High max

High temperature tolerant

Grow on low cost substrate
High qP at low
Extra cellular products

Low viscous biomass

Generally regarded as safe (GRAS)
Mineral medium

Thus, it is important to
analyze the kinetics of
growth and product
formation, designing the
optimal feed profile, role
of transport processes,
maybe use bigger
reactors.

Remember!
qS or completely
determines the microbial
behavior. It must be
controlled at an optimal
value

19

Batch, continuous, and fed batch processes

In batch reactor: and qS are at the maximum value and not
controlled.

In chemostat: is controlled at optimum

In fed batch reactor: qS is controlled.

batch

chemostat

fed
batch

20

Constant volume batch reactor

The steps:
1. Add sterile growth medium solution (substrate consist of
C, N, P source, K+, Mg+, salts, vitamin, trace elements)
and choose the right T and pH.
2. Add electron acceptors (O2, NO3-, etc)

3. Inoculate microorganism at initial time with concentration

CXo.
4. Run the fermentation and harvest the broth, proceed to the
downstream processing.

21

Constant volume batch reactor

What is the plot CS and CX with time?
Derive from the mass balance, one can obtain that the growth
is exponential, the slope of the curve CX vs. time is rX.
Parameters

State
variables

reactor

operator

micro
organism

CS0
CX0

qSmax
max
YSXmax
mS
KS

CS
CX

22

Substrate concentration profile

At constant volume:

dCS
rS qS C X
dt
For batch reactor:

dCS
max
max
maxt
qS C X qS C Xoe
dt

t = 0, CS = Cso ; CSo CS

max
qS
C
max Xo

exp(

t) 1

max

Hence, by putting CS = 0, t end of batch is obtained

The slope in CS vs. t plot is rS.
23

Try to sketch the curve CS, CX, , -qS, YSX, rX, -rS versus
time for batch reactor!

Conclusion for the batch reactor

, YSX, and qS are constant at the maximum values
Microbial model parameters are determined by combining the
exponential equation with CS and CX data versus time.
, the most important rate, can not be controlled

24

Chemostat
Steady state: constant volume, constant in and out flow rates.
Constant CS and CX.
Parameters

State
variables

reactor

operator

micro
organism

CS,in
L,in

qSmax
YSXmax
mS
KS

L,out

CS
CX

25

Steps in operating continuous culture

Sterilized reactor is filled with sterile growth medium

solution containing substrate at CS,in

Air sparging starts to provide electron acceptor

Add a small amount of microorganism (inoculum)

Start the medium in flow (often L,in and L,out are nearly
equal, not always)

Wait until steady state is achieved

The main property of chemostat:

1. CS and CX are independently manipulated by the user by
manipulating transport (to and from the reactor) of substrate
and biomass.
2. Excellent experimental tool to study microbial kinetics,
stoichiometry, under controlled conditions.

26

Biomass mass balance in chemostat

In out + gen = acc

0 C X L,out rX V 0
C X L,out C X V

L,out
V

The is chosen by manipulating exit flow rate

Hence, all other qi-values are set (stoichiometric coupling)
Does CS depend on CS,in? Use (CS) to prove it.

27

Critical dilution rate

The maximal value for D (=) attainable in the chemostat, is
achieved when the maximal CS in achieved. At critical dilution
rate, CS CS,in.

Dcrit

max CS ,in

CS ,min

K S CS ,in

max

Note: CS,in normally in order 10000 mg/l, CS,min order 1 mg/l, KS

order 10 mg/l.

Wash out
If D > Dcrit, the cell will be washed out
Then, sketch the graph CS vs. D.

28

Substrate mass balance

L,in CS ,in L,out CS ,out rSV 0
rS CS,in CS D

L,in
L,out

This equation together with Herbert-Pirt relation provide rS and

(-qS) from measured concentration, flows, and volume.
What about CX?

D CS ,in CS
rS
CX

YSX CS ,in CS
D
qS
(mS )
max
YSX

29

The application of chemostat

1. Kinetic studies, at different = D one can varies qS, CS to
obtain YSXmax, mS, qSmax, KS.
2. Physiological and genomic micro array study studies of
microorganisms under defined steady state, I.e. substrate,
electron acceptor, N-source, type of limitation for growth.

3. Waste water treatment

4. Industrial fermentation, which is not widely applied

30

Chemostat optimization
Normally, the maximal rX and rS are needed as the economic
parameter

Try to find the optimum D where drS/dD and drX/dD = 0.

Note that rS has a maximum at higher D.

Chemostat wash out dynamic

What happen when D is close to Dcrit?
What happen to CX when D = D>Dcrit?
dC X
C X D ' C X
dt

C X C X , ss exp m ax D ' t
31

Pro and cons of chemostat

(+)
Excellent experimental tool because is defined
(-)
Low biomass and product concentration
Loss of biomass in outflow
Relatively prone to be contaminated compare to batch or fed
batch reactors
Microbial selection for non-producing mutants

32

Fed batch fermentation

In batch reactor, CS and CX are high. No transport of S or X and
no control on .

In chemostat, CS and CX are low. Transport of S or X and

control on .
In fed batch reactor. Substrate transport in, not out. No biomass
transport.
Why fed batch?
1. Low CS no toxicity / osmotic problem

2. High CX high CP easier downstream processing

3. Control of ?
33

Fed batch fermentation

Start feeding
CSO
CS

Batch phase

Feeding phase under substrate

limited conditions
CS = 1 50 mg/l.

CSO 5000
20000 mg/l

time
In substrate limited feeding phase, CS is very low. Thus, one
can use the pseudo steady state condition for substrate mass
balance

34

Substrate mass balance in fed batch reactor

d VC S
S ,in CS ,in 0 rSV 0
dt
S ,in CS ,in
rS
V
Hence, in a fed batch reactor the substrate conversion (-rS) is
controlled by the operator.
Through controlled rS, is controlled.
It is obvious that the reactor volume changes with time.
However, since the change is very small, for simplicity we can
assume constant volume, constant density.

35

Rates in fed batch reactor

We start with an assumption that volume is a variable
d VC X
Biomass mass balance
rX V VC X
dt
d VC X 1

dt VC X
Pseudo steady state substrate mass balance

Product mass balance

rS s,inCs,in
qS

CX
VC X
d VC P
qP VC X
dt
d VC P 1
qP
dt VC X

36

Possible substrate feeding strategies in a fed

batch fermentation
1. Substrate input (S,inCS,in) is constant

2. is maintained at optimum that gives maximal qP or

maximal YSP.
3. Substrate input is determined by other known reactor
limitations (oxygen, heat, etc)

Apply Herbert-Pirt equation and biomass mass balance to

calculate rX and CX.

37

Biomass mass balance in fed batch reactor

Assume constant volume (DV <<)
dC X
rX
dt

Combine with Herbert-Pirt relation and eliminate rS using

its fed batch relation:
dC X
m ax S ,in CS ,in
m ax
mS C X
YSX
YSX
dt
V
This is homogeneous differential equation in CX.

38

Biomass mass balance in fed batch reactor

dC X
m ax S ,in CS ,in
m ax
mS C X
YSX
YSX
dt
V
The first explains growth, the later is about maintenance.
Initially CX is low in a fed batch reactor. By neglecting
maintenance:
dC X
m ax S ,in CS ,in
YSX
constant
dt
V
CX increases linearly with time
Later, CX increases, more and more substrate needed for
maintenance dCX/dt decreases.

End, all substrate needed for maintenance, dCX/dt = 0

CXmax

S ,inCS ,in /V
mS

39

Fed batch reactor with constant feeding rate

The analytical solution for CX(t) in feeding phase.

max

C
1

exp

Y
S
,
in
S
,
in
max
SX mS t C exp Y max m t
C X t YSX
Xf
SX
S
max
V
mS
YSX

t = time after start feeding

CXf = biomass concentration at start feeding
Note:
For t >>> 0, CX

S ,inCS ,in

V (mS )
For t <<< (YSXmax(-mS))-1,

linear growth

m ax
mS t
1 exp YSX

m ax
mS
YSX

m ax
mS t 1
exp YSX
m ax S ,in CS ,in
C X C Xf YSX

t
40

Fed batch reactor with constant feeding rate

dC X
m ax S ,in CS ,in
m ax
mS C X (t )
YSX
YSX
dt
V
rX decreases
rX t
t
C X t

- qS t

rS
C X t

sharply decreases
qS decreases

CS decreases slowly

41

Fed batch reactor with constant optimum

= opt = constant qi, Yij are all constant.
qS constant at qSopt CS constant.
Biomass concentration increases exponentially
dC X
rX opt C X
dt
All rates increases exponentially

ri t qiopt C X t
Substrate feeding rate increases exponentially

S ,inCS ,in t rS V S ,inCS ,in t f exp optt

tf = time when feeding starts
42